Project Summary/Abstract Laboratories around the world have produced tens of thousands of distinct zebrafish lines that serve as model organisms for genetic and biomedical research that applies to human genetics and diseases. Maintaining all these valuable genotypes is expensive and beyond the capacity of even the largest stock centers. One solution is the cryopreservation of zebrafish sperm, eggs and embryos, an approach that would enable the strategy of increased multi- institutional research using animal models envisioned by the NIH Division of Comparative Medicine. Zebrafish sperm and eggs have been successfully cryopreserved, but technical challenges had prevented the cryopreservation of zebrafish embryos until recent success demonstrated by nanoComposix and Prof. John Bischof at the University of Minnesota. The main challenge with zebrafish embryo cryopreservation is the large embryo size, which limits the rate at which the embryo can be externally cooled and warmed. The innovative approach used by the applicants utilizes an injected formulation of gold nanoparticles that act as ultra- efficient heaters to generate heat internally when illuminated with an infrared laser. This technology demonstrated, for the first time, the successful cooling, cryogenic stabilization and rewarming of zebrafish embryos. Further development and commercialization of this successful proof-of-concept will address a critical need for zebrafish researchers and will form the basis for other genetic preservation applications in aquaculture and human reproduction, improving reproduction for societal, environmental and biodiversity needs. This project will further optimize the zebrafish embryo laser warming reagents, protocols, and equipment developed during the Phase I contract. The equipment will be beta tested by zebrafish research centers and a commercial system will be manufactured. The system will consist of a reagent formulation composed of a cryoprotectant agent and a gold nanoparticle, optimized to provide high photothermal conversion efficiency and minimal toxicity, and will be manufactured under a ISO 13485-compliant quality system. Reagent microinjection into the embryos and freezing and laser rewarming protocols will be refined to further improve embryo survival rates and will form the basis of training materials for end users. Finally, first-generation equipment will be developed, along with testing of automated, high-throughput methods for embryo cooling. Further technology development will provide a universal high throughput platform for preserving germplasm of other vertebrate and non-vertebrate organisms.